Cigarette use has led to an epidemic of lung cancer and heart disease and is the major preventable cause of poor health in the U.S.This epidemic is driven by the desire to feel the effects of nicotine, which are likely mediated by binding of nicotine to neuronal nicotinic acetylcholine receptors (nAChRs). The mechanisms of nicotine dependence are unknown, however, we have discovered a large family of nAChRs expressed in brain. nAChRs with a.4 and (32 subunits have the highest affinity for nicotine and chronic nicotine exposure (smoking) produces an increase, or up-regulation, of these nAChRs in human smokers and animal models. We proposed that long term nicotine exposure changes the properties of nAChRs in brain, and these changes underlie the molecular mechanism of dependence. Initial exposure to nicotine activates nAChRs, but chronic exposure depresses nAChR function (desensitization). Compensatory mechanisms (up-regulation) are induced by chronic nicotine, potentiallyto maintain normal levels of neurotransmission. Removal of agonist via smoking cessation allows recovery of nAChRs, producing an excess of active nAChRs which may contribute to the physiological desire to continue smoking. To test this hypothesis we will examine the mechanism(s) by which nicotine regulates the function and expression of the high affinity a4/|32 nAChRs using tissue culture model systems and lines of genetically altered mice. We propose to investigate the underlying molecular mechanism(s) of nAChR up-regulation using an in vitro expression system in combination with genetic and biochemical methods to characterize several proteins that we have identified that interact with cx4/|32 nAChRs. We will also explore the role of nAChR desensitization in the induction of up-regulation and its effects on behaviors related to nicotine addiction by capitalizingon the effects of several mutations identified in cx4and [32subunits which have contrasting effects on receptor desensitization and may therefore alter receptor up-regulation differently.